Fig. 3.
CHIP-K30A and Hsp70-bound CHIP are conformationally distinct from the wild-type protein. (A) Graph showing the unfolding of His-CHIP wt pre-incubated with the indicated peptides based on Hsp70 (left panel) or His-CHIP wt or K30A mutant (right panel) as a function of temperature change measured by the uptake of the fluorescent dye SYPRO Orange. Shown is the means ± S.E. of mean of 3 experiments. (B) Table listing the mid-point temperature of phase transition (Tm) of each sample in (A) that was calculated by plotting the gradient of protein unfolding against the temperature gradient [-d(RFU)/dT]. (C) InstantBlue stained gel of untagged CHIP wt or K30A (left panel) digested with the protease Glu-C. FL is the full-length protein and band 1 is a cleavage product that persists in the K30A mutant. Band 2 is only observed in digests of the wt protein. Also shown is a Glu-C digest of His-CHIP wt protein in complex with wt or mutant Hsp70 peptides (right panel).